Production of adenine nucleotide translocator (ANT), novel ANT ligands and screening assays therefor

Abstract

Compositions and methods are provided for producing adenine nucleotide translocator (ANT) polypeptides and fusion proteins, including the production and use of recombinant expression constructs having a regulated promoter. ANT ligands and compositions and methods for identifying ANT ligands, agents that bind ANT and agents that interact with ANT are also disclosed.

1. A recombinant expression construct comprising at least one regulated promoter operably linked to a first nucleic acid encoding a polypeptide having at least 95% identity to a human adenine nucleotide translocator 3 (ANT3) polypeptide comprising the amino acid sequence set forth in SEQ ID NO:33, wherein said polypeptide binds an ANT ligand. 
     
     
       2. The expression construct of  claim 1  further comprising at least one additional nucleic acid sequence that regulates transcription. 
     
     
       3. The expression construct of  claim 2  wherein the additional nucleic acid sequence that regulates transcription encodes a repressor of said regulated promoter. 
     
     
       4. The expression construct of  claim 1  wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:33. 
     
     
       5. An expression construct according to  claim 1  wherein the polypeptide is expressed as a fusion protein with a polypeptide product of a second nucleic acid sequence. 
     
     
       6. The expression construct of  claim 5  wherein the polypeptide product of said second nucleic acid sequence is an enzyme. 
     
     
       7. The expression construct of  claim 5  wherein said fusion protein localizes to membranes. 
     
     
       8. The expression construct of  claim 7  wherein said membranes are mitochondrial membranes. 
     
     
       9. An expression construct according to  claim 1  wherein the adenine nucleotide translocator polypeptide is expressed as a fusion protein with at least one product of a second nucleic acid sequence encoding a polypeptide cleavable by a protease, said adenine nucleotide translocator polypeptide being separable from the fusion protein by cleavage with the protease. 
     
     
       10. A host cell comprising a recombinant expression construct according to  claim 1 . 
     
     
       11. A host cell according to  claim 10  wherein the host cell is a prokaryotic cell. 
     
     
       12. A host cell according to  claim 10  wherein the host cell is a eukaryotic cell. 
     
     
       13. The host cell of  claim 12  wherein the eukaryotic cell is selected from the group consisting of a yeast cell, an insect cell and a mammalian cell. 
     
     
       14. The host cell of  claim 13  wherein the insect cell is selected from the group consisting of an Sf9 cell and a  Trichoplusia ni  cell. 
     
     
       15. A host cell according to  claim 10  that lacks at least one isoform of an endogenous adenine nucleotide translocator. 
     
     
       16. A host cell according to  claim 10  in which expression of at least one gene encoding an endogenous adenine nucleotide translocator isoform is substantially impaired. 
     
     
       17. A recombinant expression construct comprising at least one regulated promoter operably linked to a nucleic acid molecule comprising a first nucleic acid sequence and a second nucleic acid sequence, said first nucleic acid sequence encoding a polypeptide having at least 95% identity to a human adenine nucleotide translocator 3 (ANT3) polypeptide comprising an amino acid sequence set forth in SEQ ID NO:33, wherein said polypeptide binds an ANT ligand, and wherein said polypeptide is expressed as a fusion protein with a polypeptide product of said second nucleic acid sequence. 
     
     
       18. The expression construct of  claim 17  wherein the polypeptide product of said second nucleic acid sequence is an enzyme. 
     
     
       19. The expression construct of  claim 17  wherein said fusion protein localizes to membranes. 
     
     
       20. The expression construct of  claim 19  wherein said membranes are mitochondrial membranes. 
     
     
       21. The expression construct of  claim 17  further comprising at least one additional nucleic acid sequence that regulates transcription. 
     
     
       22. The expression construct of  claim 21  wherein the additional nucleic acid sequence that regulates transcription encodes a repressor of said promoter. 
     
     
       23. The expression construct of  claim 17  wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:33. 
     
     
       24. An expression construct according to  claim 17  wherein the adenine nucleotide translocator polypeptide is expressed as a fusion protein with at least one product of a second nucleic acid sequence encoding a polypeptide cleavable by a protease, said adenine nucleotide translocator polypeptide being separable from the fusion protein by cleavage with the protease. 
     
     
       25. A host cell comprising a recombinant expression construct according to  claim 17 . 
     
     
       26. A host cell according to  claim 25  wherein the host cell is a prokaryotic cell. 
     
     
       27. A host cell according to  claim 25  wherein the host cell is a eukaryotic cell. 
     
     
       28. The host cell of  claim 27  wherein the eukaryotic cell is selected from the group consisting of a yeast cell, an insect cell and a mammalian cell. 
     
     
       29. The host cell of  claim 28  wherein the insect cell is selected from the group consisting of an Sf9 cell and a  Trichoplusia ni  cell. 
     
     
       30. A host cell according to  claim 25  that lacks at least one isoform of an endogenous adenine nucleotide translocator. 
     
     
       31. A host cell according to  claim 25  in which expression of at least one gene encoding an endogenous adenine nucleotide translocator isoform is substantially impaired. 
     
     
       32. A recombinant expression construct according to either  claim 1  or  claim 17  wherein the expression construct is a recombinant viral expression construct. 
     
     
       33. method of producing a recombinant adenine nucleotide translocator polypeptide, comprising:
 culturing a host cell infected with the recombinant viral expression construct of  claim 32  and,  
 producing an ANT polypeptide that binds an ANT ligand.  
 
     
     
       34. A method of producing a recombinant adenine nucleotide translocator polypeptide, comprising:
 culturing a host cell comprising a recombinant expression construct comprising at least one regulated promoter operably linked to a first nucleic acid encoding an adenine nucleotide translocator polypeptide, and  
 producing an ANT polypeptide that binds an ANT ligand.  
 
     
     
       35. A method of producing a recombinant adenine nucleotide translocator 3 (ANT3) polypeptide, comprising:
 culturing a host cell comprising a recombinant expression construct comprising at least one regulated promoter operably linked to a nucleic acid molecule comprising a first nucleic acid sequence and a second nucleic acid sequence, said first nucleic acid sequence encoding a polypeptide having at least 95% identity to a human ANT3 polypeptide comprising a sequence set forth in SEQ ID NO:33, wherein said polypeptide binds an ANT ligand and, wherein the polypeptide is expressed as a fusion protein with a polypeptide product of said second nucleic acid sequence.